Matchmaker™ Gold Yeast Two Hybrid System User Manual2].pdf · Matchmaker™Gold Yeast Two-Hybrid System User Manual Protocol No. PT4084-1 Clontech Laboratories, Inc. Version No

Matchmaker™ Gold Yeast Two-Hybrid System User Manual

PT4084-1 (PR912676) Cat. No. 630489Published 15 January 2009

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Matchmaker™Gold Yeast Two-Hybrid System User Manual

Protocol No. PT4084-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR912676 A Takara Bio Company

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I. Introduction & Protocol Overview ..........................................................................................4

II. List of Components ..................................................................................................................7

III. Additional Materials Required .................................................................................................8

IV. General Considerations Regarding Yeast Two-Hybrid Libraries ............................................9

V. List of Abbreviations ............................................................................................................... 10

VI. Control Experiments .............................................................................................................. 11A. General Considerations ..................................................................................................................... 11B. Protocol: Control Mating Protocol ................................................................................................... 11

VII. Cloning and Testing Bait for Autoactivation and Toxicity ................................................... 14A. Generate a Bait Clone .......................................................................................................................14B. Detecting Bait Expression .................................................................................................................15C. Protocol: Testing Your Bait for Autoactivation...................................................................................15D. Protocol: Testing Your Bait for Toxicity .............................................................................................16

VIII. Two-Hybrid Library Screening Using Yeast Mating ...........................................................17

IX. Analysis of Results .................................................................................................................21A. Too Few Positives .............................................................................................................................21B. Too Many Positives ..........................................................................................................................21

X. Confirmation of Positive Interactions & Rescue of the Prey Plasmid ................................22A. Protocol: Yeast Colony PCR to Eliminate Duplicates ........................................................................23B. Protocol: Rescue and Isolation of Library Plasmid Responsible for Activation of Reporters ...............24C. Protocol: Distinguishing Genuine Positive from False Positive Interactions .......................................25D. Sequence Analysis of a Genuine Positive ...........................................................................................27E. Biochemical Methods to Confirm Positive Interactions .....................................................................28F. Downstream Analysis ........................................................................................................................28

XI. Troubleshooting Guide ...........................................................................................................29

XII. References .............................................................................................................................31

Appendix A: Mate & Plate™ Library Construction ...................................................................33

Appendix B: Library Titering .......................................................................................................36

Appendix C: Control Plasmid Information .................................................................................37

Appendix D: Yeast Growth Media & Supplements ...................................................................38

Table of Contents

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Clontech Laboratories, Inc. www.clontech.com Protocol No. PT4084–1A Takara Bio Company Version No. PR912676

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Table of Contents continuedList of Figures

Figure 1. The two-hybrid principle. ........................................................................................................... 4Figure 2. Reporter gene constructs in Matchmaker yeast strains. ............................................................... 5Figure 3. Two-hybrid screening using Mate & Plate™ Libraries. ................................................................ 6Figure 4. Simple, one-step PCR cloning with In-Fusion Advantage PCR Cloning Kits. ..........................14Figure 5. An example of a typical yeast zygote. ........................................................................................18Figure 6. High stringency screening of potential interactors. ...................................................................20Figure 7. Strategies for analyzing and verifying putative positive interactions ...........................................22Figure 8. Illustration of the activation of reporter gene expression in genuine and false positives. ...........25Figure 9. Using cotransformation on selective media to verify protein interactions. ...............................................26Figure 10. Map of pGBKT7-53 DNA-BD control plasmid. ...................................................................37Figure 11. Map of pGADT7-T AD control plasmid. .............................................................................37

List of TablesTable I: Mating the Pretransformed Control Strains ................................................................................ 11Table II: Recommended PCR Master Mixes for PCR Amplification of Library Vector Insert ..................23Table III: Yeast Host Strain Genotypes ....................................................................................................33Table IV: Phenotype Testing on Various SD Media .................................................................................33Table V: Components of Yeast Media Set 2 & Yeast Media Set 2 Plus .....................................................38Table VI: Individual Yeast Media Pouches for Matchmaker Gold Protocols .............................................38Table VII: Additional Media & Media Supplements Required for a Two-Hybrid Screen .........................39



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I. Introduction & Protocol OverviewPrinciple of the Two-Hybrid AssayIn a Matchmaker GAL4-based two-hybrid assay, a bait protein is expressed as a fusion to the Gal4 DNA-binding do-main (DNA-BD), while libraries of prey proteins are expressed as fusions to the Gal4 activation domain (AD; Fields & Song, 1989; Chien et al., 1991). In the Matchmaker Gold Yeast Two-Hybrid System (Cat. No. 630489), when bait and library (prey) fusion proteins interact, the DNA-BD and AD are brought into proximity to activate transcription of four independent reporter genes (AUR1-C, ADE2, HIS3, and MEL1) (Figure 1).

This technology can be used to:

identify novel protein interactions•

confirm putative interactions•

define interacting domains•

Figure 1. The two-hybrid principle. Two proteins are expressed separately, one (a bait protein) fused to the Gal4 DNA-binding domain (BD) and the other (a prey protein) fused to the Gal4 transciptional activation domain (AD). In Y2HGold Yeast Strain, activation of the reporters (AUR1-C, ADE2, HIS3, and MEL1) only occurs in a cell that contains proteins which interact and bind to the Gal4-responsive promoter.

The BaitTo make your GAL4 DNA-BD/bait construct, we recommend using pGBKT7, which is available separately (Cat. No. 630443) or as a component of our Matchmaker Gold Two-Hybrid System (Cat. No. 630489). To investigate ternary protein complexes, we suggest you use pBridge (Cat. No. 630404), a three-hybrid vector that contains two MCS regions so that you can express a Gal4 DNA-BD fusion and a second protein of interest that may act as a "bridge" between bait and prey.

mRNA

GAL Promoter

RNA Pol II

5'

GAL4 DNA-BD

GAL4 AD

Prey

Bait


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I. Introduction & Protocol Overview continued

Figure 2. Reporter gene constructs in Matchmaker yeast strains. In Y2HGold, the HIS3, ADE2, and MEL1/AUR1-C reporter genes are under the control of three completely heterologous Gal4-responsive promoter elements—G1, G2, and M1, respectively. The protein-binding sites within the promoters are different, although each is related to the 17-mer consensus sequence recognized by Gal4 (Giniger et al., 1985; Giniger & Ptashne, 1988).

Four Reporter Genes to Detect Protein InteractionsThere are four integrated reporter genes under the control of three distinct Gal4-responsive promoters (Figure 2) in Clontech’s Y2HGold Yeast Strain, which are used to detect two-hybrid interactions.

AUR1-C. A dominant mutant version of the AUR1 gene that encodes the enzyme inositol phosphoryl ceramide syn-thase. AUR1-C is expressed in Y2HGold Yeast Strain in response to protein-protein interactions that bring the GAL4 transcriptional activation and DNA binding domains into close proximity. In Saccharomyces cerevisiae, its expression confers strong resistance (AbAr) to the otherwise highly toxic drug Aureobasidin A (Cat No. 630466). This drug re-porter is preferable to nutritional reporters alone, due to lower background activity. For example, the use of this reporter alone results in considerably less background than a histidine reporter alone.

HIS3. Y2HGold is unable to synthesize histidine and is therefore unable to grow on media that lack this essential amino acid. When bait and prey proteins interact, Gal4-responsive His3 expression permits the cell to biosynthesize histidine and grow on –His minimal medium.

ADE2. Y2HGold is also unable to grow on minimal media that does not contain adenine. However, when two proteins interact, Ade2 expression is activated, allowing these cells to grow on –Ade minimal medium.

MEL1. MEL-1 encodes a-galactosidase, an enzyme occurring naturally in many yeast strains. As a result of two-hybrid interactions, a-galactosidase (MEL1) is expressed and secreted by the yeast cells. Yeast colonies that express Mel1 turn blue in the presence of the chromagenic substrate X-a-Gal (Cat. Nos. 630462 & 630462).

Note: X-a-Gal is not X-Gal, and is not a substrate for b-galactosidase.

Three Different Binding SitesThree promoters controlling the four reporter genes AUR1-C, HIS3, ADE2, and MEL1 in Y2HGold are unrelated except for the short protein binding sites in the UAS region that are specifically bound by the Gal4 DNA-BD. Thus, library proteins that interact with unrelated sequences flanking or within the UAS (i.e., false positives) are automatically screened out.

NOTE

G1 Promoter lacZ

Y187 (Library Host Strain) reporter gene constructs

Y2HGold (Mating Partner) reporter gene constructs

G1 Promoter HIS3

M1 Promoter AUR1-C

G2 Promoter ADE2

M1 Promoter MEL1

M1 Promoter MEL1



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I. Introduction & Protocol Overview continued

Figure 3. Two-hybrid screening using Mate & Plate™ Libraries. Your bait protein is expressed as a fusion with the Gal4 DNA-BD in yeast strain Y2HGold. The high-complexity library, which expresses fusions with the Gal4 AD, is provided in yeast strain Y187. When cultures of the two transformed strains are mixed together overnight, they mate to create diploids. Diploid cells contain four reporter genes: HIS3, ADE2, MEL1, and AUR1-C, that are activated in response to two-hybrid interactions.

Matchmaker Screening Protocol Overview

The entire Matchmaker screening process (Figure 3) consists of the following steps:

Step 1. Perform control experiments•

Step 2. Clone and test bait for autoactivation and toxicity•

Step 3. Screen Mate & Plate™ library•

Step 4. Confirm and interpret results•

Confirm patch positiveson QDO/X/A(Section VIII)

Clone bait(Section VII.A)

Check expression(Section VII.B)

Test for autoactivationand toxicity

(Section VII.C & VII.D)

Mate with library(Section VIII)

Plate on DDO/X/Aand pick blue colonies(Section VIII)


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II. List of ComponentsMatchmaker Gold Yeast Two-Hybrid System (Cat. No. 630489)

Store all Matchmaker vectors at –20°C, all yeast (S. cerevisiae) strains at –70°C, and all Yeast Media Pouches at room temperature.

For the Yeastmaker™ Yeast Transformation System 2, store carrier DNA and control plasmid at –20°C, and all other components at room temperature.

Matchmaker Vectors

50 µl pGBKT7 DNA-BD Cloning Vector (0.1 µg/µl)•

50 µl pGADT7 AD Cloning Vector (0.1 µg/µl)•

50 µl pGBKT7-53 Control Vector (0.1 µg/µl)•

50 µl pGADT7-T Control Vector (0.1 µg/µl)•

50 µl pGBKT7-Lam Control Vector (0.1 µg/µl)•

Matchmaker Gold Yeast Two-Hybrid System User Manual (PT4084-1)•

pGBKT7 Vector Information (PT3248-5)•

pGADT7 AD Vector Information (PT3249-5)•

Matchmaker Yeast Strains

0.5 ml Y2HGold Yeast Strain •

0.5 ml Y187 Yeast Strain •

Yeastmaker™ Yeast Transformation System 2 (also available separately as Cat No. 630439)

2 x 1 ml Yeastmaker Carrier DNA, denatured (10 mg/ml)•

20 µl pGBT9 (0.1 µg/µl; positive control plasmid)•

2 x 50 ml 50% PEG•

50 ml 1 M LiAc (10X)•

50 ml 10X TE Buffer•

50 ml YPD Plus Liquid Medium•

Yeastmaker Yeast Transformation System 2 User Manual (PT1172-1)•

Complimentary Yeast Media Pouches

1 x 0.5 L YPDA•

1 x 0.5 L YPDA with Agar•

1 x 0.5 L SD–Leu with Agar•

1 x 0.5 L SD–Trp with Agar•



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III. Additional Materials RequiredA. Mate & Plate™ Libraries (several catalog items; see www.clontech.com for current list)

Store all Mate and Plate Libraries and Control vials at –70°C.

5 x 1.0 ml Library Aliquots (Universal Human and Mouse are also sold as 2 x 1 ml vials) •

1 x 1.0 ml Mate & Plate Control•

Alternatively, you can make your own Mate & Plate library using our Make Your Own “Mate & Plate” Library System (Cat. No. 630490).

B. Accessory KitsYeastmaker Yeast Transformation System 2• (supplied with your system—see Section II; also sold separately as Cat. No. 630439)

Easy Yeast Plasmid Isolation Kit• (50 preps; Cat. No. 630467)

C. Tools for Plating YeastTools for plating yeast include a sterile glass rod—and a bent Pasteur pipette or 5 mm glass beads for spread-•ing cells on plates. (Use 5–7 beads per 100 mm plate, or 15–20 beads for a 150 cm plate).

D. Yeast MediaTable V (in Appendix D) lists the components of the Yeast Media Set 2 (Cat. No. 630494) and the Yeast Media Set 2 Plus (Cat. No. 630495). These media sets contain a complete assortment of mixes for preparing eight specialized broth and agar media, designed for use with the Matchmaker Gold Yeast Two-Hybrid System, in convenient, “ready-mixed” foil pouches. The Yeast Media Set 2 Plus also contains the additional media supplements Aureobasidin A and X-a-Gal, which are required for the protocols described in this user manual. Table VI (in Appendix D) contains information for purchasing each of the media mixes separately, in packs of 10 pouches, and Table VII (in Appendix D) contains preparation instructions for all additional required media supplements and information for purchasing Aureobasidin A and X-a-Gal separately.

Additionally, the following should be considered when culturing yeast for a two-hybrid screen:

See Appendix D for working and stock concentrations of Aureobasidin A and X-• a-Gal.

SD medium• (synthetically defined medium) is minimal media that is routinely used for culturing S. cerevi-siae. SD base supplies everything that a yeast cell needs to survive (including carbon and nitrogen sources). Essential amino acids, which are added to SD base to create minimal medium, are already included premixed in Clontech’s Yeast Media Pouches. The particular minimal medium that is chosen will determine which plasmids and/or activated reporters are selected for.

SD/–Leu/–Trp• dropout supplement is used to select for the bait and prey plasmids. SD/–Leu/–Trp dropout is so called because the medium includes every essential amino acid except for leucine and tryptophan, which are omitted from the formulation (or “Dropped Out”). Cells harboring Matchmaker bait and prey plasmids are able to grow because the plasmids encode tryptophan and leucine biosynthesis genes, respectively, that are otherwise absent from the cell. We often refer to SD/–Leu/–Trp as Double Dropout (DDO) in this user manual.

SD/–Ade/–His/–Leu/–Trp• dropout supplement is used to select for the bait and prey plasmids, and in addition, for the activation of the Gal-responsive HIS3 and ADE2 genes as part of the confirmation step of the two-hybrid assay. Colonies that grow on this Quadruple Dropout (QDO) contain both bait and prey plasmids and also express proteins that interact with each other to activate HIS3 and ADE2. This medium is used at the end of the two-hybrid screen to confirm interactions.


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IV. General Considerations Regarding Yeast Two-Hybrid LibrariesUse of Mate & Plate™ LibrariesPlease note that the protocols described in this manual assume that you are using a Mate & Plate™ Library. These libraries utilize the natural ability of haploid yeast strains such as Y187 and Y2HGold to mate with each other to form a diploid cell, providing a very easy way to introduce an entire library (prey) to your bait.

Clontech • strongly recommends using Mate & Plate Libraries with the Matchmaker Gold System. These libraries provide by far the simplest method for yeast two-hybrid screening because no library-scale transfor-mations or labor-intensive amplifications are needed. Thus, very little optimization and hands-on time are required.

Several Mate & Plate libraries are available for purchase from Clontech, supplied as 5 x 1 ml vials (and also •sold as 2 x 1 ml vials for Universal Human and Universal Mouse libraries). Alternatively you can easily “Make Your Own Mate & Plate Library” using Cat No. 630490, and store enough 1 ml vials for more than 100 library screens.

A single 1ml Mate & Plate Library aliquot is sufficient for each complete library screening (>1x10• 6 indepen-dent clones).

See Appendix A for more details on prey vectors used to construct Mate & Plate Libraries. These libraries •are supplied in Saccharomyces cerevisiae strain Y187, in Freezing Medium. Depending on which library you purchase, your library may be cloned into pGADT7-Rec2, pGADT7-RecAB, or pACT2. (See Certificate of Analysis for details.). All are compatible with Matchmaker Gold.

A Mate & Plate Control is supplied with all Mate & Plate libraries. This control is Y187 Yeast Strain •pretransformed with our pGADT7-T positive control plasmid, which expresses the Gal4 AD-SV40 large T-antigen fusion protein. See Section VI for control experiments.

Once a library aliquot has been thawed, do not refreeze it. With every freeze/thaw cycle, there is a ~10% loss •in viability, which can affect the quality of the library.

The recommended Freezing Medium consists of YPDA broth + 25% glycerol (see Appendix D).•



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V. List of AbbreviationsAD fusion library A cDNA library (such as a Mate & Plate Library) constructed in an [or AD library] activation domain (AD) vector such that the proteins encoded by the inserts are fused to the 3’ end of the Gal4 AD

AD/library plasmid Plasmid encoding a fusion of the Gal4 activation domain and a library cDNA

AD/library protein A protein fusion comprised of the Gal4 activation domain and a polypeptide encoded by a library cDNA

AD vector Plasmid encoding the yeast Gal4 activation domain

DNA-BD vector Plasmid encoding the Gal4 DNA-binding domain

DNA-BD/bait plasmid Plasmid encoding a fusion of the Gal4 DNA binding domain and a bait cDNA

DNA-BD/bait protein A protein fusion comprised of the Gal4 DNA binding domain and a polypeptide [or “bait”] encoded by a bait cDNA

Yeast PhenotypesAbA The antibiotic Aureobasidin A, which is toxic to yeast at low concentrations (0.1–0.5 µg/ml). It acts by inhibiting a yeast enzyme, inositol phosphoryl ceramide synthase.

AbAr Resistance to the antibiotic Aureobasidin A, conveyed by expression of the AUR1-C gene product.

AUR1-C A dominant mutant version of the AUR1 gene, which encodes the enzyme inositol phosphoryl ceramide synthase. This gene is expressed in Yeast Strain Y2HGold, in response to protein-protein interactions that bring the GAL4 activation and binding domains in proximity, thus conferring resistance to the antibiotic Aureobasidin A.

Ade–, or His–, or Requires adenine (Ade), or histidine (His) or leucine (Leu), or tryptophan (Trp) in the Leu–, or Trp– medium to grow; i.e., is auxotrophic for one (or more) of these specific nutrients

LacZ+ Expresses the LacZ reporter gene; i.e., is positive for ß-galactosidase (ß-gal) activity.

Mel1+ Expresses the MEL1 reporter gene; i.e., is positive for a-galactosidase (a-gal) activity

MiscellaneousSD Minimal, synthetically defined medium for yeast; is comprised of a nitrogen base, a carbon source (glucose unless stated otherwise), and a DO supplement

DO Dropout (supplement or solution); a mixture of specific amino acids and nucleosides used to supplement SD base to make SD medium; DO solutions are missing one or more of the nutrients required by untransformed yeast to grow on SD medium

DDO Double dropout medium: SD/–Leu/–Trp

DDO/X/A Double dropout medium: SD/–Leu/–Trp supplemented with X-a-Gal and Aureobasidin A

TDO Triple dropout medium: SD/–His/–Leu/–Trp or SD/–Ade/–Leu/–Trp

QDO Quadruple dropout medium: SD/–Ade/–His/–Leu/–Trp

QDO/X/A Quadruple dropout medium: SD/–Ade/–His/–Leu/–Trp supplemented with X-a-Gal and Aureobasidin A

YPD A blend of yeast extract, peptone, and dextrose in optimal proportions for growth of most strains of S. cerevisiae

YPDA YPD medium supplemented with adenine hemisulfate (1X concentration = 120 µg/ml)


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VI. Control Experiments

Please read the entire Protocol before starting Use this procedure to perform a control mating before screening a two-hybrid library.

A. General ConsiderationsTo familiarize yourself with the procedures and expected results of a two-hybrid assay, use these procedures to perform a control mating before you begin screening the library. Select for diploids and for two-hybrid protein-protein interac-tors as described below.

pGBKT7-53 encodes the Gal4 DNA-BD fused with murine p53; pGADT7-T encodes the Gal4 AD fused •with SV40 large T-antigen. Since p53 and large T-antigen are known to interact in a yeast two-hybrid assay (Li & Fields, 1993; Iwabuchi et al., 1993), mating Y2HGold [pGBKT7-53] with Y187 [pGADT7-T] will result in diploid cells containing both plasmids that can activate all four reporters (Table I).

A negative control should also be performed using pGBKT7-Lam (which encodes the Gal4 BD fused with •lamin) and pGADT7-T. Diploid yeast containing pGBKT7-Lam and pGADT7-T will grow on SD/–Leu, SD/–Trp and SD/–Leu/–Trp (DDO) minimal media, but no colonies should grow on DDO + AbA.

Table I indicates the selection media required for strains containing a DNA-BD vector, AD vector, or both, •as well as the selection for diploids expressing interacting proteins.

Table I: Mating the Pretransformed Control Strains

Mating Strain [plasmid]Plate on SD Minimal Agar Medium Selects for

MEL1 Phenotype

Y2HGold [pGBKT7-53]

x

Y187[pGADT7-T]

–Leu pGADT7-T White

–Trp pGBKT7-53 White

–Leu/–Trp1 (DDO)Diploids containing pGBKT7-53 and pGADT7-T

White

–Leu/–Trp/X-a-Gal/AbA2 (DDO/X/A)

Diploids that have also activated Aureobasidin A resistance and a-galactosidase through protein-protein interactions

Blue

1 Controls for mating efficiency.2 Selects for diploids expressing interacting proteins.

B. Protocol: Control Mating Protocol1. Materials:

SD/–Trp with Agar (see Appendix D)•

SD/–Leu with Agar (see Appendix D)•

SD/–Trp/X-• a-Gal agar plates (see Appendix D)

SD/–Leu/–Trp agar plates (see Appendix D)•

SD/–Leu/–Trp/X-• a-Gal/AbA agar plates (see Appendix D)

2 x YPDA Broth (see Appendix D)•

YPDA Broth + 25% glycerol (Freezing Medium; see Appendix D)•

Y2HGold Yeast Strain (Bait Strain)•

Y187 Yeast Strain (Prey Strain)•

pGBKT7-53 Positive Control Bait Plasmid•

pGBKT7-Lam Negative Control Bait Plasmid•

pGADT7-T Positive Control Prey Plasmid•

Protocol7–10 days



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NOTES:

Use the Yeastmaker Yeast Transformation System 2 (supplied with this system) for all transformations. •

X-• a-Gal is not the same as X-Gal.

Use the Yeastmaker Yeast Transformation System 2 according to the small-scale protocol in the accompanying 2. user manual to perform the following three transformations.

Strain Transformation Plasmid Plating Medium

Y2HGold pGBKT7-53 SD/–Trp with Agar

Y2HGold pGBKT7-Lam SD/–Trp with Agar

Y187 pGADT7-T SD/–Leu with Agar

Grow at 30°C for 3 days.3.

Note: If you wish, you may stop the experiment at this step and resume work later. The plates can be stored at 4ºC in subdued lighting for up to one month.

Pick one 2–3 mm colony of each type for use with the following small-scale mating procedure (Steps 5–7).4.

Positive Control Mating:• Y2HGold [pGBKT7-53] and Y187 [pGADT7-T]

Negative Control Mating:• Y2HGold [pGBKT7] and Y187 [pGADT7]

Place both colonies in a single 1.5 ml centrifuge tube containing 500 µl of 2X YPDA and vortex to mix.5.

Incubate with shaking at 200 rpm at 30°C overnight [20–24 hr].6.

From the mated culture (0.5 ml), spread 100 µl of 1/10, 1/100, and 1/1,000 dilutions on each of the follow-7. ing agar plates. Incubate plates (colony side facing downward) at 30°C for 3–5 days.

SD/–Trp•

SD/–Leu•

SD/–Leu/–Trp (=DDO)•

SD/–Leu/–Trp/X-• a-Gal/AbA (=DDO/X/A)

Expected results after 3–5 days:8.

Positive control:

Similar number of colonies on DDO and DDO/X/A agar plates•

Colonies on DDO/X/A are blue•

Negative control:

Colonies on DDO, but no colonies on DDO/X/A agar plates•

Notes:

For positive interactions, theoretically, the number of colonies should be the same on both media. DDO se-•lects for the presence of both plasmids (i.e, proper mated diploids) and DDO/X/A selects for the plasmids as well as for the interactions of the hybrid proteins encoded by them to activate the AbAr and MEL1 reporters. However, a difference (approximately 10–20% lower on DDO/X/A) is usually observed.

If you see no colonies on DDO, compare to colony counts on SD/–Trp and SD–/Leu single dropout media to •

determine if there was a problem with the bait or the prey cultures, respectively.

NOTE

Attention

NOTE

VI. Control Experiments continued


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VI. Control Experiments continued

Pick healthy 2 mm colonies from DDO plates, restreak onto fresh DDO plates, and incubate at 309. °C for 3–4 days.

Short-term storage (< 4 weeks): Seal with Parafilm® and store at 4°C.•

Long-term storage: Scoop a large healthy colony and fully resuspend in 500 µl of YPDA Broth + 25% •glycerol (Appendix D). Store at –80°C.

Notes:

These diploids are useful as reference strains for checking new batches of growth media, and for compari-•sons in future experiments.

When reviving frozen stocks, remember to restreak onto DDO selective medium.•

NOTE



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VII. Cloning and Testing Bait for Autoactivation and Toxicity

Please read the entire Protocol before starting detailed instructions are provided to test your bait for autoactivation (section c) and toxicity (section d).

A. Generate a Bait CloneGenerate a GAL4 DNA-BD fusion by cloning your gene of interest in frame with the GAL4 DNA binding domain of the bait plasmid pGBKT7 (see Appendix C and www.clontech.com for map).

For an extremely simple cloning procedure, we recommend using one of Clontech’s In-Fusion™ Advantage® PCR Cloning Kits (see www.clontech.com for details).

PCR Cloning of Your Bait into pGBKT7The following method describes a simple and highly efficient method to clone your gene in-frame with the GAL4BD in pGBKT7. The magic of In-Fusion Advantage (Cat. Nos. 639201, 639202, 639206 & 639207) means that:

Your bait is automatically cloned in-frame with the bait.•

Virtually every clone contains your insert.•

It does not matter what sites are present on your bait sequence since for In-Fusion • cloning you do not digest it.

1. Digest pGBKT7 to completion with BamHI and EcoRI, then spin column-purify.

2. Amplify your bait insert by PCR using oligos that contain a 24 bp homology to your bait, and a 15 bp homology to the linear ends of pGBKT7, which are designed as follows:

Forward Primer (111 = first codon of your bait)5’-C ATG GAG GCC GAATTC 111 222 333 444 555 666 777 888

Reverse Primer (LLL = reverse complement of last codon of your bait)5’-GC AGGTCGACGGATCC LLL NNN NNN NNN NNN NNN NNN NNN

NOTE: These primers actually contain 16 bp of homology in order to keep the BamHI and EcoRI sites intact.

3. Mix the bait and linear pGBKT7 together and “fuse”’ using the In-Fusion enzyme.

See the In-Fusion Advantage PCR Cloning Kit User Manual (PT4065-1) at www.clontech.com for additional details regarding PCR cloning procedures.

Figure 4. Simple, one-step PCR cloning with In-Fusion Advantage PCR Cloning Kits.

x x

Recombinantvector

Single-tube protocol


15

Matchmaker™ Gold Yeast Two-Hybrid System User ManualMatchmaker™Gold Yeast Two-Hybrid System User Manual

VII. Cloning and Testing Bait for Autoactivation and Toxicity continuedIf you wish to find proteins that interact with a membrane-bound or secreted protein, it may be necessary to first •modify the protein (van Aelst et al., 1993) or to use only selected domains as the bait (as in Kuo et al., 1997).

In order to confirm that the fusion construct is in-frame, the fusion junction may be sequenced using a standard •T7 primer.

B. Detecting Bait ExpressionIf you wish to determine whether or not your bait is expressed well in yeast, both of the following antibodies will detect bait proteins in yeast containing pGBKT7-based bait plasmids (via Western blot). In order to make yeast protein extracts (yeast cannot simply be boiled or sonicated to extract protein), we strongly recommend that you use the supporting protocols provided at www.clontech.com

GAL4 DNA-BD Monoclonal Antibody (Cat. No. 630403)•

c-Myc Monoclonal Antibody (Cat. No. 631206)•

Note: Use Y2HGold [pGBKT7-53] as a positive control that expresses a 57 kD protein.

C. Protocol: Testing Your Bait for AutoactivationAs a first step for any two-hybrid screen, it is imperative to confirm that your bait does not autonomously activate the reporter genes in Y2HGold, in the absence of a prey protein.

Materials:1.

pGBKT7 containing your gene of interest cloned in frame with the • GAL4 DNA-BD (pGBKT7).

Competent Y2HGold cells [see Yeastmaker™ Yeast Transformation System 2 User Manual (PT1172-1), •supplied with this system]

SD/–Trp/ X-• a-Gal agar plates (Appendix D)

SD/–Trp/X-• a-Gal/AbA agar plates (Appendix D)

NOTE: X-a-Gal is required, not X-Gal (Appendix D).

Transform 100 ng of your pGBKT7+Bait construct using the Yeastmaker Yeast Transformation System 2 (supplied 2. with this system).

NOTE: (POSITIVE and NEGATIVE CONTROLS) For comparison, we recommend that you also plate the diploid controls that you created in Section VI; but plate them on DDO/X/A since they contain both bait and prey plasmids).

Spread 100 µl of a 1/10 dilution and a 1/100 dilution of your transformation mixture onto separate plates, as 3. follows:

SD/–Trp plates = SDO•

SD/–Trp/ X-• a-Gal = SDO/X plates

SD/–Trp/X-• a-Gal/AbA = SDO/X/A plates

Expected results after 3–5 days:4.

Sample Selective Agar Plate Distinct 2 mm Colonies Color

Bait autoactivation test SDO Yes White

Bait autoactivation test SDO/X Yes White or Very Pale Blue

Bait autoactivation test SDO/X/A No N/A

Positive control (Section VI) DDO/X/A Yes Blue

NOTE

Protocol4–6

days.

Attention



16

VII. Cloning and Testing Bait for Autoactivation and Toxicity continued

NOTE: If your bait autoactivates the AbAr reporter (i.e., blue colonies appear on SDO/X/A) check to see if it also activates the His3/Ade2 reporters by plating on SD–Trp/–His/–Ade. See Section XI (Troubleshooting) if your bait activates all the reporters.

D. Protocol: Testing Your Bait for ToxicityYou should demonstrate that your bait protein is not toxic when expressed in yeast. If your bait is toxic to the yeast cells, both solid and liquid cultures will grow more slowly.

If expression of your bait protein does have toxic effects, you may wish to switch to a vector (such as pGBT9) that has a lower level of expression.

NOTE: pGBT9 is supplied as a transformation control in Clontech's Yeastmaker Transformation System 2 (supplied with this system).

Materials:1.

Y2HGold competent cells [see Yeastmaker™ Yeast Transformation System 2 User Manual (PT1172-1), •supplied with this system]

SD/–Trp agar plates (Appendix D)•

SD/–Trp broth (Appendix D)•

Transform 100 ng of the following vectors:2.

pGBKT7 (empty)•

pGBKT7 + cloned bait gene•

Spread 100 µl of 1/10 and 1/100 dilutions of your transformation mixtures onto SD/–Trp.3.

Grow at 304. °C for 3–5 days:

NOTE: If your bait is toxic, you may notice that colonies containing your bait vector are significantly smaller than colonies containing the empty pGBKT7 vector.

Protocol4–6

days.

NOTE


17


VIII. Two-Hybrid Library Screening Using Yeast Mating

Please read the entire Protocol before starting detailed instructions are provided for performing a yeast two-hybrid library screening.

Simply mix a concentrated bait culture with 1 ml of your Mate & Plate library and incubate overnight before •plating on DDO/X/A selective media (see Figure 3).

In addition to plating on DDO/X/A media, it is imperative that you determine the number of clones that •you screened on DDO (following the protocol below ensures this). Screening fewer than 1 million clones may result in an inability to detect positive interactions

With a “normalized” Mate & Plate library, up to threefold fewer clones need to be screened, because gene representation has been equalized, significantly reducing the abundance of housekeeping genes in the library. Several normalized libraries are available from www.clontech.com.

1. Materials:

Mate & Plate™ Library—make your own, using our • Make Your Own “Mate & Plate” Library System (Cat. No. 630490) or purchase separately.

Bait construct transformed into Y2HGold on SD/–Trp (Section VII)•

SD/–Trp Broth (Appendix D)•

2X YPDA Broth (Appendix D)•

0.5X YPDA broth (Appendix D)•

kanamycin sulfate (50 mg/ml)•

YPDA + 25% glycerol [Freezing Medium] (Appendix D)•

The following selective SD agar plates (also see Appendix D):•

Agar Media Acronym Number of Plates

SD/–Trp — 5–10 (100 mm plates)

SD/–Leu — 5–10 (100 mm plates)

SD/–Leu/–Trp DDO 5–10 (100 mm plates)

SD/–Leu/–Trp/X-a-Gal/AbA DDO/X/A 50–55 (150 mm plates)

SD/–Ade/–His/–Leu/–Trp/X-a-Gal/AbA QDO/X/A 5—10 (100 mm plates)

NOTE: Clontech’s Mate & Plate libraries are supplied in strain Y187, so your bait must be in strain Y2HGold.

If you wish to “Make your Own Mate & Plate Library” use our kit (Cat. No. 630490)

(see www.clontech.com for details)

Protocol5–6

days

NOTE



18

VIII. Two-Hybrid Library Screening Using Yeast Mating continuedConstruct your bait, test for autoactivation and toxicity (Section VII).2.

Perform the control experiments (Section VI).3.

NOTE: Control experiments are strongly recommended; the control strains will aid interpretation of results when you screen your library.

Prepare a concentrated overnight culture of the bait strain (Y2HGold [pGBKT7+Bait]) as follows: 4.

Inoculate one fresh, large (2–3 mm) colony of your bait strain into 50 ml of SD/–Trp liquid medium.a.

Incubate shaking (250–270 rpm) at 30°C until the ODb. 600 reaches 0.8 (16–20 hr).

Centrifuge to pellet the cells (1,000 g for 5 min), discard the supernatant.c.

Resuspend the pellet to a cell density of >1x10d. 8 cells per ml in SD/–Trp (4–5 ml). [The cells can be counted using a hemocytometer.]

Combine the Library Strain with the Bait Strain as follows:5.

Thaw a 1 ml aliquot of your library strain in a room temperature water bath. Remove 10 µl for titering on a. 100 mm SD/–Leu agar plates (see Appendix B, Section B for library titering instructions).

NOTE: Use a hemocytometer to count the cells. Your 1 ml library aliquot should contain >2x107 cells. To check the titer, spread 100 ml of 1/100, 1/1,000, 1/10,000 dilutions on SD/–Leu agar plates. If your titer is 2x107 cells/ml, you will obtain 200 colonies on the 1/10,000 dilution plate.

Combine 1 ml of your Mate & Plate Library with 4–5 ml Bait Strain (from Step 4) in a sterile 2 L flask.b.

Add 45 ml of 2xYPDA liquid medium (with 50 µg/ml kanamycin).c.

Rinse cells from the library vial twice with 1 ml 2xYPDA and add to the 2 L flask.d.

Incubate at 306. °C for 20–24 hr, slowly shaking (30–50 rpm).

IMPORTANT: Use the lowest shaking speed possible that prevents the cells from settling at the base of the flask. Vigorous shaking can reduce the mating efficiency, but shaking too slowly will cause the cells to sedi-ment, also lowering the mating efficiency.

NOTE

NOTE

Attention

After 20 hr, check a drop of the culture under a 7. phase contrast microscope (40X). If zygotes are present, continue to Step 8, if not, allow mating to continue; incubate for an additional 4 hr.

NOTE: A zygote typically has a 3-lobed structure (see Figure 5). The lobes represent the two hap-loid parental cells and the budding diploid cell. Some zygotes may resemble a clover leaf, while others may take on a shape similar to a “Mickey Mouse” face.

Centrifuge to pellet the cells (1,000 g for 10 min).8.

NOTE

Figure 5. An example of a typical yeast zygote.


19


VIII. Two-Hybrid Library Screening Using Yeast Mating continuedMeanwhile, rinse the 2L flask twice with 50 ml 0.5xYPDA (with 50 µg/ml kanamycin), combine the rinses, 9. and use this to resuspend the pelleted cells.

Centrifuge to pellet the cells (1,000 g for 10 min) and discard the supernatant.10.

Resuspend all pelleteted cells in 10 ml of 0.5X YPDA/Kan liquid medium. Measure the total volume of cells 11. + medium.

NOTE: e.g., 10 ml medium + 1.5 ml cells = 11.5 ml

From the mated culture, spread 100 µl of 1/10, 1/100, 1/1,000, and 1/10,000 dilutions on each of the fol-12. lowing 100 mm agar plates and incubate at 30°C for 3–5 days.

SD/–Trp•

SD/–Leu•

SD/–Leu/–Trp (DDO)•

NOTE: This step is essential to calculate the number of clones screened (see Step 14).

Plate the remainder of the culture, 200 µl per 150 mm on DDO/X/A (50–55 plates). Incubate at 30°C for 13. 3–5 days.

Calculate the number of screened clones14. (diploids) by counting the colonies from the DDO plates after 3–5 days.

Number of Screened Clones = cfu/ml of diploids x resuspension volume (ml)•

It is imperative that at least 1 million diploids are screened, since using less than this will result in less •chance of detecting genuine interactions on Aureobasidin A plates (DDO/X/A).

Example Calculation

Resuspension volume (Step 11) = 11.5 ml •

Plating Volume = 100 µl•

50 colonies grew on the 1/1,000 dilution on DDO plates.•

Therefore Number of Clones screened = 50 x 11.5 x 10 x 1,000 = 5.75 million

15. Determine the Mating Efficiency.

Mating efficiencies of 2–5% are readily achieved using this procedure. If your mating efficiency is less than 2% and you cannot screen 1 million diploids (Step 14), refer to the Troubleshooting Guide (Section XI) for tips on improving the mating efficiency, and screen more clones.

Measure viabilitiesa.

No. of cfu/ml on SD/–Leu = viability of the Prey Library •

No. of cfu/ml on SD/–Trp = viability of Bait •

No. of cfu/ml on SD/–Leu/–Trp = viability of diploids•

NOTE



20

VIII. Two-Hybrid Library Screening Using Yeast Mating continued

NOTE: The strain (bait or prey) with the lower viability is the "limiting partner."

Calculate Mating Efficiency (percentage of diploids):b.

No. of cfu/ml of diploids x 100 = % Diploids No. of cfu/ml of limiting partner

Example Calculation

Resuspension volume (Step 11) = 11.5 ml •

Plating Volume = 100 µl•

5,000 colonies grew on the 1/10,000 on SD/–Trp•

100 colonies grew on the 1/10,000 dilution on SD/–Leu•

50 colonies grew on the 1/1,000 dilution on DDO plates•

Therefore (in cfu/ml),

Viability of Prey Library = 1 x 10 – 7

Viability of Bait = 5 x 10 – 8

Viability of Diploid = 5 x 10 – 5

Since the Prey Library is the limiting partner in this example, mating efficiency is calculated as follows:

5 x 105 x 100 = 5% Mating Efficiency1 x 107

Patch out all the blue colonies that grew on DDO/X/A onto higher stringency QDO/X/A agar plates using a 16. flat sterile toothpick or yellow pipette tip (Figure 6).

NOTE

NOTE: Although it is possible to screen directly on high stringency QDO/X/A, Clontech recommends screening first on lower stringency DDO/X/A to detect as many positives interactors as possible before confirming those by patching on highest stringency plates.

All QDO/X/A positive interactions must be further analyzed (Section X) to identify duplicates and to verify 17. that the interactions are genuine.

NOTE

Figure 6. High stringency screening of potential interactors.

DDO/X/A QDO/X/A


21


IX. Analysis of ResultsAfter high-stringency patching to identify potential binding partners for your protein of interest, you may have very few positives, or too many positives to analyze. In these scenarios, we recommend checking the following:

A. Too Few Positives Have you screened >1 million independent clones? Refer to Section VIII, Step 14 to determine if you screened 1 mil-lion independent clones? Optimize the mating/transformation procedure (see Section XI. Troubleshooting Guide) and repeat the screening procedure.

Check that your DDO/X/A and QDO/X/A growth media performs as expected with the positive and nega-•tive controls. Very little AbA (70 ng/ml) is required, so make certain not to use too much.

If you screened >1 million independent clones and detected no positive colonies on DDO/X/A , repeat the •screen with a reduced concentration of Aureobasidin A (50 ng/ml instead of 70 ng/ml):

B. Too Many Positives Have you determined that your bait does not autoactivate the reporters (Section VII.C)?

Check that your DDO/X/A and QDO/X/A growth media performs as expected with the positive and nega-•tive controls.

Your bait may interact with a partner that is abundant in the library. Sort duplicates by Yeast Colony PCR •(Section X.A). After the clones have been sorted into groups, a representative of each unique type can then be analyzed for false positive interactions (Section X.C).

Alternatively, you may wish to try a Normalized Mate & Plate Library; see • www.clontech.com for details.



22

X. Confirmation of Positive Interactions & Rescue of the Prey Plasmid

Please read the entire Protocol before starting detailed instructions are provided for yeast colony Pcr to eliminate duplicates (section a), rescue and isolation of library plasmids responsible for activation of reporters (section b), and distinguishing genuine positive from false positive interactions (section c).

The following represents the recommended order of events to confirm that the positive interactions are genuine. The strategy is summarized in Figure 7. Note, however, that your preferred order of events may be somewhat determined by the number of positives obtained from your assay. For instance, if your bait protein interacts with a protein that is abundant in the library, you may have a large number of potential positives to sort, many of which may be the same. In this case you may choose to perform colony PCR (Section X.A) to sort the duplicate clones before segregating and rescuing the plasmid. If you have a low number of positive clones, you may choose to omit the colony PCR screening step altogether and proceed directly to the Easy Yeast Plasmid Isolation Kit (Cat. No. 630467)

We recommend performing the following steps prior to sequencing your positive clones:

Yeast Colony PCR•

Rescue and isolation of the library plasmid responsible for activation of reporters•

Distinguishing genuine positive from false positive interactions•

Figure 7. Strategies for analyzing and verifying putative positive interactions

Confirm Interaction in Yeast Cotransform DNA-BD/bait and positive AD/library plasmids

into Y2HGold

Confirm + phenotype by streaking on

high-stringency QDO/X/A

Transform plasmids into E. coli and purify DNA

Confirm protein interactionsin mammalian cells

Mammalian Two-HybridAssay Kit 2

(Cat. No. 630305)

Matchmaker Chemiluminescent Co-IP

Kit (Cat. No. 630459)

Additional Two-Hybrid Tests- Switch Vectors- Frameshift Mutations- Site-specific mutations/deletions

Sequence cDNA inserts

Eliminate duplicates containing the same AD/library plasmid by yeast colony PCR

Rescue plasmids from yeast usingthe Easy Yeast Plasmid Isolation Kit

(Cat. No. 630467)


23


X. Confirmation of Positive Interactions & Rescue of the Prey Plasmid continued

A. Protocol: Yeast Colony PCR to Eliminate DuplicatesThis procedure uses the Matchmaker AD LD-Insert Screening Amplimer Set (Cat. No 630433) and Advantage® 2 PCR Polymerase Mix (Cat. No. 639201). We strongly recommend using the Advantage 2 Polymerase Mix, rather than any other DNA polymerase formulation, because we find that it performs well in yeast cell samples.

Materials1.

Single colonies of yeast from a two-hybrid screen, growing on QDO/X/A plates (Appendix D)•

0.8% TAE Agarose/EtBr gel•

Master mix using Advantage 2 Polymerase, as described below.•

LD insert, screening amplimer (Cat. No. 630433), Advantage 2 (Cat. No. 639201)•

Prepare a PCR master mix by combining the components specified in Table II.2.

Table II: Recommended PCR Master Mixes for PCR Amplification of Library Vector Insert

Reagent 1 rxn 10 rxns + 1 extra 25 rxns + 1 extra

PCR-grade deionized H2O 41 µl 451 µl 1,066 µl

10X Advantage 2 PCR Buffer 5 µl 55 µl 130 µl

5' AD LD Amplimer Primer (20 µM) 1 µl 11 µl 26 µl

3' AD LD Amplimer Primer (20 µM) 1 µl 11 µl 26 µl

50X dNTP Mix (10 mM each) 1 µl 11 µl 26 µl

50X Advantage 2 Polymerase Mix 1 µl 11 µl 26 µl

Total 50 µl 550 µl 1,300 µl

Prealiquot 50 µl of PCR mix into tubes or wells and keep on ice. Then, using a pipette tip, scrape a few cells 3. from a colony into an individual tube or well and pipette up and down to mix the cells. Test 50 colonies.

TIP: Using too many cells can inhibit the PCR reaction. Simply touching the colony with the tip should provide a sufficient quantity of cells. If your PCR mix turns turbid, you may be using too many cells.Attention

Follow the following PCR cycling parameters:4.

94• °C for 3 min

25–30 cycles•

94°C for 30 sec

68°C for 3 min

Analyze PCR products by electrophoresis on a 0.8% TAE Agarose/EtBr gel.5.

Load 5 µl per lane.•

The presence of more than a single band is common, indicating the presence of more than one prey •plasmid in a cell (see Section X.B).

NOTE: To confirm that similar sized bands contain the same insert, you may choose to digest the PCR product with AluI or HaeIII or other frequently cutting enzymes and electrophorese on a 2% agarose/EtBr gel.

Protocol4 hr–1 day

NOTE



24

X. Confirmation of Positive Interactions & Rescue of the Prey Plasmid continuedIf a high percentage of the colonies appear to contain the same AD/library insert, expand your PCR analysis 6. to another batch of 50 colonies.

At this stage, to quickly identify the clones, the PCR products (observed as a single band on gel) can be spin 7. column-purified and sequenced using a standard T7 primer.

B. Protocol: Rescue and Isolation of Library Plasmid Responsible for Activation of Reporters

1. Segregation of Library Plasmid in YeastTransformed yeast cells (unlike transformed E.coli cells) can harbor more than one version of a related plasmid. This means that in addition to containing a prey vector that expresses a protein responsible for activating the reporters, a yeast cell may also contain one or more prey plasmids that do not express an interacting protein.

If you rescue the plasmid via • E.coli transformation without first segregating the non-interacting prey, there is a chance that you will rescue a non-interacting prey plasmid.

To increase the chance of rescuing the positive prey plasmid, we recommend that you streak 2–3 times on •DDO/X (no Aureobasidin A), each time picking a single blue colony for restreaking. After the first streak-ing, you may see a mixture of blue and white colonies, indicating segregation of positive interactors (blue) from non-interactors (white). After streaking one or two more times, you should only see blue colonies. The plasmid should be rescued from one of these clones (see Step 2).

2. Rescuing the Library Plasmid from YeastThe following methods are recommended for rescuing your plasmid from yeast:

To identify the gene responsible for the positive interaction, rescue the plasmid from yeast cells grown on •QDO/X using the Easy Yeast Plasmid Isolation Kit (Cat. No. 630467)

If your bait is cloned in pGBKT7• (which contains a kanamycin resistance gene), you can select for the prey plasmid simply by selection on LB plus 100 µg/ml ampicillin using any commonly used cloning strain of E.coli (e.g. DH5a™, or Fusion-Blue™ from Clontech).

If your bait is cloned into an older Matchmaker bait vector• such as pGBT9, pAS2 or pAS2-1, both bait and prey vectors confer ampicillin resistance. To select for colonies that contain only the prey vector, you can trans-form KC8 E.coli cells (Cat. No. 630435) and plate on M9 minimal medium lacking leucine. KC8 cells have a defect in leuB that can be complemented by yeast Leu2. Note that the same dropout supplements used for yeast SD medium can be used for M9 minimal medium e.g., the –Leu DO supplement (Cat. No. 630417).

Protocol2–7

days


25



C. Protocol: Distinguishing Genuine Positive from False Positive InteractionsY2HGold Yeast Strain contains four reporters under the control of three distinct GAL4 UAS sequences. As a result, of following the high stringency screening protocols described in this user manual, the incidence of false positives is reduced to a minimum compared to other systems. The incidence of false positives is further reduced with Normalized Mate & Plate Libraries due to more equal representation of each transcript. However, with every two-hybrid screen there is a chance of detecting false positives and it is important to confirm that your interactions are genuine using the following criteria (see Figure 8):

Genuine Positive• : Both Bait and Prey are required to activate the Gal4-responsive reporters

False Positive• : Prey can activate the Gal4-responsive reporters in the absence of your bait.

Protocol4–6

days

Figure 8. Illustration of the activation of reporter gene expression in genuine and false positives.

You can confirm protein interactions in yeast on selective media (see Appendix D for recipes) using the following cotrans-formation procedure (Figure 9). This can also be done by yeast mating (see Section VIII).

Materials:1.

Competent Y2HGold cells [see Yeastmaker™ Yeast Transformation System 2 User Manual (PT1172-•1), supplied with this system]

SD/-Leu/-Trp/X-• a-Gal Agar (Appendix D) = DDO/X

SD/-Ade/-His/-Leu/-Trp/ X-• a-Gal /AbA (Appendix D) = QDO/X/A

Using the small-scale transformation procedure, cotransform100 ng of each of the following pairs of vectors 2. into Y2HGold Competent Cells:

pGBKT7/Bait + Prey (in pGADT7, pGADT7-Rec, or pGADT7-RecAB)•

Empty pGBKT7 + Prey (in pGADT7, pGADT7-Rec, or pGADT7-RecAB)•

NOTE: We recommend that you perform the experiment side by side with the positive and negative controls (Section VI).NOTE

Promoter Reporter Gene


DNA BD

AD

AD

Prey


Prey

Bait Prey

AD

Genuine Positive

Prey Alone: No activation

False Positive

Prey Alone: Activation

Bait + Prey Positive interaction



26


Spread 100 µl of 1/10 and 1/100 dilutions of the transformation mix on the following plates:3.

DDO/X•

QDO/X/A•

Expected results after 3–5 days at 304. °C (Figure 9):

Genuine Positive:a.


Bait + candidate prey DDO/X Yes Blue

QDO/X/A Yes Blue

Empty pGBKT7 + candidate prey DDO/X Yes White

QDO/X/A No N/A

False Positive: b.

For false positive interactions, similar numbers of blue colonies are observed on all plates (indicating that the prey does not require your bait to activate the reporters).


Bait + candidate prey DDO/X Yes Blue

QDO/X/A Yes Blue

Empty pGBKT7 + candidate prey DDO/X Yes Blue

QDO/X/A Yes Blue

NOTE: Theoretically, for positive interactions, the number of colonies should be the same on both media: DDO selects for both plasmids, and QDO/X/A selects for the plasmids as well as for the interaction of the hybrid proteins encoded by them. However a difference is usually observed (10–60% lower on QDO/X/A, depending on the strength of the interaction).NOTE

1. Cotransform:

2. Plate on:

(b) QDO/X/A

(a) DDO/X

(a) pGBKT7/Bait +

candidate prey

(b) Empty pGBKT7 +

candidate prey

Figure 9. Using cotransformation on selective media to verify protein interactions. Expected results from genuine interactions.


27


X. Confirmation of Positive Interactions & Rescue of the Prey Plasmid continuedD. Sequence Analysis of a Genuine Positive

Once an interaction has been verified as being genuine, the prey insert can be identified by sequencing. Use only DNA isolated from E.coli for this procedure. AD/library cDNA inserts can be sequenced using the following:

Matchmaker AD LD-Insert Screening Amplimer Set (Cat. No. 630433),•

T7 Sequencing Primer•

Always check the vector sequence against the primer you wish to use. Be aware that some Matchmaker AD plasmids (e.g., pACT2) do not contain a T7 Promoter.

Verify the presence of an open reading frame (ORF) fused in frame to the GAL4 AD sequence, and compare the sequence to those in GenBank, EMBL, or other databases.

NOTES:

Before considering any of the following possibilities, we recommend that you verify that your clone is not a false positive (Section X.C).

Most genuine positive clones will activate all reporters, however it is possible that •some library clones only activate a selection of the reporters, for example the colony grows on QDO but does not turn blue in the presence of X-a-Gal. This may be due to inaccessibility of a particular prey fusion protein to a specific UAS. Confirm the inter-action by additional means such as coimmunoprecipitation

Most library clones will contain some 3’ untranslated region, be sure to scan the en-•tire sequence to find any portion of coding region fused in-frame to the GAL4 AD.

Yeast tolerate translational frameshifts. A large ORF in the wrong reading frame •may correspond to the protein responsible for the interaction. To verify this, re-clone the insert in-frame (this can be easily done using Clontech’s In-Fusion PCR Cloning Systems (see www.clontech.com) and determine if the AbAr, ADE2, HIS3, and MEL1 reporters are still active if your bait is also present.

If your sequencing results reveal a very short peptide (<10 amino acids) fused to the •AD—or no fusion peptide at all—keep sequencing beyond the stop codon. You may find another (larger) open reading frame (ORF). Such gaps can occur when a portion of the 5' untranslated region of an mRNA is cloned along with the coding region. A Western blot using Gal4 AD Antibody (Cat. No.630402) will reveal the presence and size of an AD fusion protein.

In some cases, two different ORFs may be expressed as a fusion with the AD even •though a non-translated gap comes between them. This is due to occasional transla-tional read-through.

If your sequencing results fail to reveal any ORF in frame with the AD coding region, •it could be that the positive library clone is transcribed in the reverse orientation from a cryptic promoter within the ADH1 terminator on the bait plasmid (Chien et al., 1991).

NOTE



28


E. Biochemical Methods to Confirm Positive InteractionsWe recommend confirming positive interactions using the following methods:

After sequencing the positive clones, most researchers choose to confirm each protein-protein interaction •using independent, biochemical methods, such as affinity chromatography and/or immunoprecipitation (Fields & Sternglanz, 1994). For this type of analysis, we recommend the Matchmaker Chemiluminescent Co-IP Kit (Cat. No. 630459) which allows you to identify true physical protein interactions by a co-immunoprecipitation assay from mammalian cell extracts.

You can also test protein-protein interactions in mammalian cells using either the • Matchmaker Mam-malian Two-Hybrid Assay Kit 2 (Cat. No. 630305) or the pCMV-Myc & pCMV-HA Vector Set (Cat. No. 631604) The Vector Set includes c-Myc and HA-Tag antibodies for the isolation and identification of protein-protein complexes.

F. Downstream AnalysisYou may wish to compare the strengths of two different interactions—for example, between a bait and two different prey proteins; or analyze the effects of point mutations on the strength of interaction, using the following methods:

Quantitative test for interactions• : The Gal-responsive LacZ gene (b-galactosidase) integrated in Y187 is not secreted (in contrast to a-galactosidase encoded by MEL1) and it cannot be used for blue/white screening on agar plates. However, LacZ is an ideal reporter for quantitative studies of protein-protein in-teractions. We recommend the use of strain Y187 for such quantitative studies because the LacZ promoter in this strain expresses strongly (Y2HGold/Y187 diploids can also be used). Quantitative LacZ assays are described in supporting Matchmaker protocols at www.clontech.com.

Confirm interactions via immunoprecipitation assays• : Clontech offers the Matchmaker Chemilu-minescent Co-IP System to perform simple immunoprecipitation assays.The Matchmaker Chemilu-minescent Co-IP Vector Set (Cat. No. 630458) combines the bright fluorescence of our Living Colors AcGFPl tag with the sensitive chemiluminescent detection capability of our ProLabel technology. The vector set offers you two mammalian expression vectors—pAcGFP1-C and pProLabel-C—for genera-tion of N-terminal-tagged AcGFP1-bait and ProLabel prey fusion proteins. After the AcGFP1-tagged bait and ProLabel-tagged prey fusion proteins are coexpressed in mammalian cells, use our Matchmaker Chemiluminescent Co-IP Kit (Cat. No. 630459) to perform immunoprecipitation.


29


PROBLEM CAUSE SOLUTION

DNA-BD/bait activates reporter genes

The bait protein has a transcriptional activation domain. This is espe-cially likely if the bait protein is a transcription factor (Ma & Ptashne, 1987; Ruden et al., 1991; Ruden, 1992). Acidic amphipathic domains are often responsible for unwanted transcriptional activation (Ruden et al., 1991; Ruden, 1992).

Remove the activating domain by creating specific deletions within the gene. Retest the deletion con-structs for activation. At the amino acid level, a net negative charge per 10 amino acids is a minimal AD. Note that such deletions may also eliminate a potentially interacting domain.

Excessive background Improper media preparation• Remake SD/–Leu/–Trp/X-a-Gal/AbA medium.

0.5X YPDA medium is too rich for •the resuspension of transformed cells

Use water, TE, or 0.9% NaCl.

Low mating efficiency Insufficient number of pretrans-formed bait cells in the mating

When you prepare the overnight liquid culture of the bait strain, be sure to use a large, fresh colony for the inoculum. After centrifuging and resuspend-ing the culture, count the cells using a hemacytom-eter. The concentration should be ≥1 x 108 cells/ml, an ~100-fold excess over the pretransformed library cells.

One or both of the hybrid proteins is toxic to yeast

You may be able to genetically engineer the hybrid protein in a way that will alleviate its toxicity but still allow the interaction to occur; or use a DNA-BD or AD vector that expresses lower levels of the fu-sion protein (e.g., pBridge or pGBT9).

Bait protein is toxic to the yeast cells In some cases, strains that do not grow •well in liquid culture will grow reasonably well on agar plates. Resuspend the colony in 1 ml of SD/–Trp, then spread the cell suspension on five 100-mm SD/–Trp plates. Incubate the plates at 30°C until the colonies are confluent. Scrape the colonies from each plate, pool them in one tube, and resuspend in a total of 5 ml of 0.5X YPDA. Use the cell suspension in the normal mating procedure.

It may be necessary to perform the mating •on agar plates (Bendixen et al., 1994) or on filters (Fromont-Racine et al., 1997). Be sure to set up controls that will allow you to com-pare the library mating efficiency with that of your bait strain mated to Y187[pGADT7-T] and with that of Y187[pGADT7-T] mated to Y2HGold[pGBKT7-53].

Bait proteins may interfere with mating •if they are highly homologous to proteins involved in yeast mating (e.g., pheromone receptors). If sequence information on your bait protein is available, check it for homol-ogy to proteins known to be involved in yeast mating (Schultz et al., 1995; Pringle et al., 1997). In the rare case of homology to a pheromone receptor, it may be necessary to screen the library using a conventional library-scale yeast transformation.

XI. Troubleshooting Guide



30

PROBLEM CAUSE SOLUTION

Failure to detect known protein interactions

If one of the following situations •is occurring, it may interfere with the ability of the two-hybrid proteins to interact: (1) the hybrid proteins are not stably expressed in the host cell; (2) the fused GAL4 domains occlude the site of interaction; (3) the hybrid protein folds improperly; or (4) the hybrid protein cannot be localized to the yeast nucleus. (See van Aelst et al. [1993] for one example.)

Construct hybrids containing different domains of the bait protein. For example, to study proteins that normally do not localize to the nucleus, it may be necessary to generate mutant forms of the protein that can be transported across the nuclear membrane.

Some types of protein interac-•tions may not be detectable in a GAL4-based system.

Try using a LexA-based two-hybrid system.

Some protein interactions are not •detectable using any type of two-hybrid assay.

AD/library plasmid activates all four reporters independent of the DNA-BD/bait

False positives. The AD/library hybrid activates transcription and binds because it recognizes the promoter regions of the reporters.

Refer to Section X for methods to verify protein interactions; see Serebriiskii et al. (2000) and Bartel et al. (1993a) for further discussion of false posi-tives.

XI.Troubleshooting Guide continued


31


XII. References• Forgeneralreviewsonyeasttwo-hybridsystems,seeAllenet al., 1995; Bartel et al., 1993a, 1993b; Bartel & Fields,

(1997); Fields, 1993; Fields & Sternglanz, 1994; Fritz & Green, 1992; Guarente, 1993; Hopkin, 1996; Luban & Goff, 1995; McNabb & Guarente, 1996; Mendelsohn & Brent, 1994.

• AnextensivelistofMatchmakerSystemcitationscanbeobtainedfromourwebsite(www.clontech.com).

• Foradditionaltwo-hybridreferences,seetheGolemislabWebSite(http://www.fccc.edu:80/research/labs/golemis)oruse MedLine (http://www.ncbi.nlm.nih.gov/PubMed/medline.html) and search under key words "two-hybrid.”

Allen, J. B., Wallberg, M. W., Edwards, M. C. & Elledge, S. J. (1995) Finding prospective partners in the library: the yeast two-hybrid system and phage display find a match. TIBS 20:511–516.

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.)

Bartel, P. L. & Fields, S. (1997) The Yeast Two-Hybrid System (Oxford University Press, Oxford).

Bartel, P. L., Chien, C.-T., Sternglanz, R. & Fields, S. (1993a) Using the two-hybrid system to detect protein-protein interactions. In Cellular Interactions in Development: A Practical Approach., ed. Hartley, D. A. (Oxford University Press, Oxford) pp. 153–179.

Bartel, P. L, Chien, C.-T., Sternglanz, R. & Fields, S. (1993b) Elimination of false positives that arise in using the two-hybrid system. BioTechniques 14:920–924.

Bendixen, C., Gangloff, S. & Rothstein, R. (1994) A yeast mating-selection scheme for detection of protein-protein interactions. Nucleic Acids Res. 22:1778–1779.

Borson, N. D., Sato, W. L. & Drewes, L. R. (1992) A lock-docking oligo(dT) primer for 5’ and 3’ RACE PCR. PCR Methods Appl. 2:144–148.

Chenchik, A., Diatchenko, L., Chang, C. & Kuchibhatla, S. (1994). Great Lengths cDNA Synthesis Kit for high yields of full-length cDNA. Clontechniques IX(1):9–12.

Chien, C. T., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Nat. Acad. Sci. USA 88:9578–9582.

Fields, S. (1993) The two-hybrid system to detect protein-protein interactions. METHODS: A Companion to Meth. Enzymol. 5:116–124.

Fields, S. & Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340: 245–247.

Fields, S. & Sternglanz, R. (1994) The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 10: 286–292.

Fritz, C. C. & Green, M. R. (1992) Fishing for partners. Current Biol. 2:403–405.

Fromont-Racine, M., Rain, J.-C. & Legrain, P. (1997) Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nature Genetics 16:277–282.

Giniger, E., Varnum, S. M. & Ptashne, M. (1985) Specific DNA binding GAL4, a positive regulatory protein of yeast. Cell 40:767–774.

Giniger, E. & Ptashne, M. (1988) Cooperative DNA binding of the yeast transcriptional activator GAL4. Proc. Natl. Acad. Sci. USA 85:382–386.

Golemis, E. A., Gyuris, J. & Brent, R. (1996) Analysis of protein interactions; and Interaction trap/two-hybrid systems to identify interacting proteins. In Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), Sections 20.0 and 20.1.

Guarente, L. (1993) Strategies for the identification of interacting proteins. Proc. Natl. Acad. Sci. USA 90:1639–1641.

Gubler, U. & Hoffman, B. J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263–269.

Guthrie, C. & Fink, G. R. (1991) Guide to yeast genetics and molecular biology. In Methods in Enzymology (Academic Press, San Diego)194:1–932.

Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. & Elledge, S. J. (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805–816.

Hopkin, K. (1996) Yeast two-hybrid systems: more than bait and fish. J. NIH Res. 8:27–29.

Iwabuchi, K., Li, B., Bartel, P. & Fields, S. (1993) Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. Oncogene 8:1693–1696.

Kuo, H. J., Maslen, C. L., Keene, D. R. & Glanville, R. W. (1997) Type VI collagen anchors endothelial basement membranes by interacting with type IV collagen. J. Biol. Chem. 272: 26522–26529.

Li, B. & Fields, S. (1993) Identification of mutations in p53 that affect its binding to SV40 T antigen by using the yeast two-hybrid system. FASEB J. 7:957–963.

Luban, J. & Goff, S. P. (1995) The yeast two-hybrid system for studying protein-protein interactions. Current Opinion in Biotechnol. 6:59–64.

McNabb, D. S. & Guarente, L. (1996) Genetic and biochemical probes for protein-protein interactions. Curr. Opin. Biotechnol. 7(5):554–559.



32

XII. References continuedMendelsohn, A. R. & Brent, R. (1994) Biotechnology applications of interaction traps/two-hybrid systems. Curr. Opinion in Biotechnol. 5:482–486.

Moqadam, F. & Siebert, P. (1994) Rapid Amplification of the 3' end of cDNAs with the 3'-AmpliFINDER RACE Kit. Clontechniques IX(3):6–9.

Pringle, J. R., Roach, J. R. & Jones, E. W., eds. (1997) The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology (Cold Spring Harbor Laboratory Press, Cold Spring Habor, NY).

Ruden, D. M. (1992) Activating regions of yeast transcription factors must have both acidic and hydrophobic amino acids. Chromosoma 101:342–348.

Ruden, D. M., Ma, J., Li, Y., Wood, K. & Ptashne, M. (1991) Generating yeast transcriptional activators containing no yeast protein sequences. Nature 350:250–251.

Schultz, J., Ferguson, B. & Sprague, Jr., G. F. (1995) Signal transduction and growth control in yeast. Curr. Opinion in Genet. Devel. 5:31–37.

Serebriiskii, I., Estojak J., Berman M. & Golemis E. A. (2000) Approaches to detecting false positives in yeast two-hybrid systems. Biotechniques 28:328–336.

van Aelst, L., Barr, M., Marcus, S., Polverino, A. & Wigler, M. (1993) Complex formation between RAS and RAF and other protein kinases. Proc. Natl. Acad. Sci. USA 90:6213–6217.


33


Appendix A: Mate & Plate™ Library ConstructionA Host Strain InformationThe phenotypes and complete genotypes of Y2HGold and Y187 (the library strain) are shown in Tables III and IV. For additional information on the growth and maintenance of yeast, see the supporting Matchmaker protocols at www.clontech.com. We also recommend the Guide to Yeast Genetics and Molecular Biology (Guthrie & Fink, 1991).

Table III: Yeast Host Strain Genotypes

Strain Genotypea ReportersTransformation

Markers Reference

Y2HGoldb, d MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ,LYS2 : : GAL1UAS–Gal1TATA–His3, GAL2UAS–Gal2TATA–Ade2URA3 : : MEL1UAS–Mel1TATA

AUR1-C MEL1

AbAr, HIS3, ADE2, MEL1

trp1, leu2 Nguyen,unpublished

Y187c MATa, ura3-52, his3-200,ade2-101, trp1-901, leu2-3, 112,gal4Δ, gal80Δ, met–,URA3 : : GAL1UAS–Gal1TATA–LacZ,MEL1

MEL1, LacZ trp1, leu2 Harperet al., 1993

a The GAL1, GAL2, and MEL1 upstream activating sequences (UASs) are recognized and bound by the Gal4 BD. The trp1, his3, gal4, and gal80 mutations are all deletions; leu2-3, 112 is a double mutation.

b Y2HGold is a derivative of strain PJ69-2A (James et al., 1996). The ade2-101 mutation in the precursor strain, PJ69-2A, was replaced (by recombination) with the GAL2–Ade2 reporter construct. In the absence of GAL4, Y2HGold displays the Ade– phenotype.

c The LacZ reporter construct was integrated into the yeast genome by homologous recombination at the ura3-52 mutation (A. Holtz, unpublished). Recombinants were selected on SD/–Ura. The met– phenotype in this strain is unstable.

d The AUR1-C reporter construct was integrated into the yeast genome by homologous recombination at the ura3-52 mutation (Y. Nguyen, unpublished). Recombinants were selected on SD/–Ura. The met– phenotype in this strain is unstable.

Table IV: Phenotype Testing on Various SD Media

Strain SD/–Ade SD/–His SD/–Leu SD/–Trp SD/–Ura

Y2HGold – – – – +

Y187 – – – – +

Y2HGold [pGBKT7-53] – – – + +

Y187 [pGADT7-T] – – + – +

Control Diploid1 + + + + +

1 Diploid strain derived from mating Y2HGold [pGBKT7-53] with Y187 [pGADT7-T].

B General Considerations for Mate & Plate Library ConstructionMatchmaker Libraries may be cloned into one of several commonly used Gal4 AD vectors. Three of these are described below. To find out which vector your library was constructed in, refer to the Certificate of Analysis (CofA) included with your library. Vector maps are available at www.clontech.com.

Libraries constructed in pGADT7-Rec•

Mate & Plate Libraries cloned in pGADT7-Rec are constructed using our Make Your Own “Mate & Plate” Li-brary System (Cat. No. 630490). pGADT7-Rec offers a simple and efficient method for constructing two-hy-brid libraries via recombination-mediated cloning in vivo directly in S. cerevisiae. See the Make Your Own “Mate & Plate” Library System User Manual (PT4085-1) at www.clontech.com for a description of the procedure.



34

Appendix A: Mate & Plate™ Library Construction continuedLibraries constructed in pGADT7-RecAB•

Normalized Mate & Plate libraries are constructed in pGADT7-RecAB. cDNA synthesized using our SMART™ technology is normalized to reduce the proportion of highly abundant transcripts. Normalized cDNA is SfiI-digested, and cloned into the SfiI A/B sites of pGADT7-RecAB.

Once cloned, the library is amplified in E. coli, rescued, and used to transform yeast strain Y187. The result-ing colonies are harvested at high density in freezing medium and immediately aliquoted and frozen at –70°C.

Libraries Constructed in pACT2•

Mate & Plate Libraries that are cloned in pACT2 were prepared using a modified Gubler & Hoffman proce-dure (1983). Once cloned, the library is amplified in E. coli. The library plasmid is then isolated and used to transform yeast strain Y187. The resulting colonies are harvested, aliquoted, and frozen at –70°C.

C. Library PrimingMatchmaker cDNA libraries are oligo(dT)-primed, or oligo(dT) + random-primed, as stated on the Certificate of Analysis (CofA); please refer to your CofA for the specific type of oligo(dT) primer used in the first-strand synthesis. The “lock-docking” oligo(dT)25d(A/C/G) primer contains a degenerate nucleotide site that positions the primer at the junction of the poly-A tail and the transcript proper (Borson et al., 1992). This primer eliminates synthesis of lengthy poly(dT) regions and thereby enhances the representation of full-length clones and 3' ends in the library (Chenchik et al., 1994; Borson et al., 1992; Moqadam & Siebert, 1994). Random priming may lead to a greater representation of all portions of the gene, including amino-terminal and internal domains, regardless of mRNA secondary structure; random priming also generates a wider size-range of cDNA. For oligo(dT) + random-primed libraries, separate first-strand syntheses are performed with each type of primer; after second-strand synthesis (before ligation to the adaptor), the cDNAs are pooled in roughly equal proportions.

Unidirectional libraries are made with oligo(dT) primers that have one vector-compatible restriction enzyme site. The other site is added (with sticky ends) by the adaptor that is ligated to the cDNA. Thus, digestion with one restriction enzyme ensures the cDNA’s proper orientation when ligated to a vector that has been digested with the appropriate two enzymes.

D. Adaptors and Linkers Please refer to the Product Certificate of Analysis (CofA) for information on the adaptor or linker used in the con-struction of your Matchmaker Library. pACT2-based libraries are made using adaptor ligation. pGADT7-Rec- and pGADT7-RecAB-based libraries are constructed using Clontech’s SMART technology, as outlined in the user manual of the ”Make Your Own Mate & Plate Library System” (Cat. No. 630490)

NOTES:

The open reading frame of the insert starts at the codon immediately following the C-terminal codon •(amino acid 881) of the GAL4 AD, not within the adaptor.

If an EcoRI linker is used, the cDNA is methylated to protect any internal EcoRI sites. •

If an adaptor is used in the construction of nondirectionally cloned libraries, no methylation or restriction •enzyme digestion of the cDNA is required; therefore, any internal EcoR I sites present in the cDNA will not be cut.

If an adaptor is used in the construction of unidirectionally cloned libraries, the cDNA is methylated to •protect the alternative site.

If the library is synthesized using EcoR I/Not I/Sal I adaptors, you may excise the inserts from the vector •using sites within the adaptor.

For information about pGADT7-Rec, please refer to Vector Information Packet PT3530-5, supplied with all •libraries constructed in this vector.

NOTE


35


Appendix A: Pretransformed Yeast Library Construction continued

E. cDNA Size FractionationAll ds cDNA is size-fractionated to remove unincorporated primers,unligated adaptors, and adaptor dimers; this process also removes low-molecular-weight (i.e., <400 bp) incomplete cDNAs. Matchmaker Libraries have a wide range of insert sizes (generally >400 bp), which may be an advantage in a two-hybrid library screening (Fritz & Green, 1992; Fields & Sternglanz, 1994). See the CofA for quality control information about insert size.

F. Normalized Mate & Plate LibrariesPlease refer to the CofA for information on the normalization procedure used in the construction of the Normalized Libraries

G. Insert Size Range and Average Insert SizeSizes are determined by running the cDNA on a gel prior to cloning, and comparing the profile to MW size markers.

H. Library Amplification Unless otherwise stated on the CofA, libraries constructed in pACT2 and pGADT7-RecAB were amplified once in E. coli. Libraries constructed in pGADT7-Rec are not amplified since they are made directly in yeast.

I. Quality Control of the Original cDNA LibrarycDNA size range and average cDNA size• are determined by running the cDNA on a gel prior to cloning and comparing the profile to MW size markers. The average insert size range is 0.5 kb to 4.0 kb.

• Number of independent clones is estimated before amplification. All libraries are guaranteed to have at least 1 x 106 independent clones and are representative of the mRNA population complexity. See Ausubel et al. (1994) for a general discussion of library size.

J. Quality Control for the Mate & Plate LibrariesRefer to the CofA included with your library.



36

Appendix B: Library TiteringA. General Considerations:

Diluted libraries are always less stable than undiluted libraries. Therefore, once the library dilutions are made, •plate them within the next hour, before misleading reductions in titer can occur.

Use proper sterile technique when aliquoting and handling libraries.•

Design and use appropriate controls to test for cross-contamination.•

Always use the recommended concentration of antibiotic (i.e., kanamycin) in the medium to suppress •growth of contaminating bacteria.

B. Library TiteringMaterials1.

YPDA Broth (Appendix D)•

SD/–Leu (100 mm plates) (Appendix D)•

Sterile glass spreading rod, bent Pasteur pipette, or 5 mm glass beads for spreading culture on plates.•

Transfer the 10 µl library aliquot (reserved from Section VIII) to 1 ml of YPDA Broth in a 1.5 ml microcen-2. trifuge tube. Mix by gentle vortexing. This is Dilution A (dilution factor = 10–2).

NOTE: Matchmaker Gold Libraries are extremely viscous, so pay special attention during aliquoting.

Remove 10 µl from Dilution A, and add it to 1 ml of YPDA Broth in a 1.5 ml microcentrifuge tube. Mix by 3. gentle vortexing. This is Dilution B (dilution factor = 10–4).

Add 10 µl from Dilution A to 50 µl of YPDA Broth in a 1.5 ml microcentrifuge tube. Mix by gentle vortex-4. ing. Spread the entire mixture onto an SD/–Leu plate.

Remove 50 µl aliquots from Dilution B and spread onto separate SD/–Leu plates as above.5.

Invert the plates and incubate at 30°C for 3–5 days. 6.

NOTE: Colony size will vary, depending on the insert.

Count the number of colonies on plates having 30–300 colonies. 7.

Calculate the titer (cfu/ml) as follows: 8.

Number of colonies = cfu/mlplating volume (ml) x dilution factor

NOTE: Due to slight variability in pipettes and pipetting techniques, a 2–5-fold range in titer calculations is not unusual.

Example calculation:•

No. of colonies on plate = 100 •

Plating volume = 0.05 ml •

Dilution factor = 10• –4

100 = 2 x 107 cfu/ml 0.05 ml x 10–4

Attention

Protocol4–6

days

NOTE


37


Appendix C: Control Plasmid Information

pGADT7-T10.0 kb

Ampr

2 mori

GAL4 AD

pUCori

PADH1

PT7

SV40 NLS

HA epitope tag

LEU2

TADH1

SV40 large T-antigen

a.a. 87

MCS a.a. 708

Figure 11. Map of pGADT7-T AD control plasmid. pGADT7-T is a positive control plasmid that encodes a fusion of the SV40 large T anti-gen (a.a. 87–708) and the GAL4 AD (a.a. 768–881). The SV40 large T antigen cDNA (GenBank Locus SV4CG), which was derived from the plasmid referenced in Li & Fields (1993), was cloned into pGADT7; plasmid modification was performed at Clontech. pGADT7-T has not been sequenced and it is not known whether any of the sites are unique.

Nde I Nco I Sfi I EcoR ISma IXma IBamH ISal IPst I

Murine p53 insert

bp 371(a.a. 72)

stop codonbp 1328 (a.a. 390)

bp 1345 pGBKT7-53

8.3 kb

Kanr

2 mori

TRP1

PADH1GAL4

DNA-BDPT7

pUCori

TT7 & ADH1

= c-Myc epitope tag

MCSHind III

(738)Hind III

(6544)

Hind III(1606)

f1ori

∆

∆

Figure 10. Map of pGBKT7-53 DNA-BD control plasmid. pGBKT7-53 is a positive control plasmid that encodes a fusion of the murine p53 protein (a.a. 72–390) and the GAL4 DNA-BD (a.a. 1–147). The murine p53 cDNA (GenBank Accession No. K01700) was cloned into pGBKT7 at the EcoR I and BamH I sites. The p53 insert was derived from the plasmid described in Iwabuchi et al. (1993); plasmid modification was performed at Clontech. pGBKT7-53 has not been sequenced.



38

Appendix D: Yeast Growth Media & SupplementsA. Ready-to-go Media Pouches Available from Clontech

Clontech offers media sets with a complete assortment of mixes in convenient, “ready-mixed” foil pouches, for use with the Matchmaker Gold Yeast Two-Hybrid System. We also provide the media supplements Aureobasidin A and X-a-Gal in an optional combination with the media set. In addition, each of the media mixes as well as these two supplements may be purchased separately.

See Table V for a list of the components of the • Yeast Media Set 2 (Cat. No. 630494) and the Yeast Media Set 2 Plus (Cat. No. 630495). The Yeast Media Set 2 Plus also contains the additional media supplements Aureobasidin A and X-a-Gal, which are required for the protocols described in this user manual.

See Table VI for information for purchasing each of the media mixes separately, in packs of 10 pouches, •

See Table VII for information on preparing all additional required media supplements and purchasing Aure-•obasidin A and X-a-Gal separately.

Table V: Components of Yeast Media Set 2 & Yeast Media Set 2 Plus

Media Pouch Quantity of Pouches Supplied Volume of Media Each Pouch Makes

YPDA Broth 2 0.5 L

YPDA with Agar 1 0.5 L

SD/–Leu Broth 1 0.5 L

SD/–Leu with Agar 1 0.5 L

SD/–Trp Broth 1 0.5 L

SD/–Trp with Agar 1 0.5 L

SD/–Leu/–Trp with Agar 10 0.5 L

SD/–Ade/–His/–Leu/–Trp with Agar 1 0.5 L

Additional Components in Yeast Media Set 2 Plus

X-alpha-Gal 250 mg —

Aureobasidin A 1 mg —

Table VI: Individual Yeast Media Pouches for Matchmaker Gold Protocols

Yeast Media Pouches Clontech Cat. No.

Quantity of Pouches Supplied

Volume of Media Each Pouch Makes.

Rich Media (for Routine Culturing of Untransformed Yeast)

YPDA Broth

YPDA with Agar

630306

630307

10

10

0.5 L

0.5 L

Minimal Media Single Dropouts (SDO)

SD–Trp Broth

SD–Trp with Agar

SD–Leu Broth

SD–Leu with Agar

630308

630309

630310

630311

10

10

10

10

0.5 L

0.5 L

0.5 L

0.5 L

Minimal Media Double Dropouts (DDO)

SD–Leu/–Trp Broth

SD–Leu/–Trp with Agar

630316

630317

10

10

0.5 L

0.5 L

Minimal Media Triple Dropouts (TDO)

SD–His/–Leu/–Trp Broth

SD–His/–Leu/–Trp with Agar

630318

630319

10

10

0.5 L

0.5 L

Minimal Media Quadruple Dropouts (QDO)

SD–Ade/–His/–Leu/–Trp Broth

SD–Ade/–His/–Leu/–Trp with Agar

630322

630323

10

10

0.5 L

0.5 L


39


Appendix D: Yeast Growth Media & Supplements continued

Table VII: Additional Media & Media Supplements Required for a Two-Hybrid Screen

Freezing Medium Preparation Instructions

YPD Medium & 25% glycerol see Section E of Appendix D

Supplement Name Clontech Cat. No. 1 Stock Solution Concentration

Aureobasidin A 630466 500 µg/ml (see Section F of Appendix D)

X-a-Gal (250 mg) 630463 20 mg/ml in dimethyl formamide

Kanamycin sulfate — 50 mg/ml stock solution

Dimethyl formamide — —

1 Unless otherwise specified

B. General Media Preparation InstructionsPrepare media by dissolving pouch contents in 500 ml ddH• 20, autoclave for 15 min at 121˚C, and allow to cool before use (or filter-sterilize broth media). Do not over-autoclave.

This media does not usually require pH adjustment, but if your source water is particularly acidic, you may •need to adjust the pH of the media to 5.8.

For additional information on preparing media from the pouches, please see the Clontech Yeast Media •Protocol-at-a-Glance (PT4057-2) at www.clontech.com

C. 2X YPDA BrothReconstitute one YPDA Broth pouch in 250 ml ddH2O and sterilize.

D. 0.5X YPDA BrothReconstitute one YPDA Broth pouch in 1 L ddH2O and sterilize.

E. Freezing MediumMix 100 ml YPDA (sterile) and 50 ml 75% glycerol (sterile).

F. Aureobasidin A Stock Solution Recipe:Dissolve 1 mg Aureobasidin A (Cat. No. 630466) in 2 ml of absolute ethanol for a stock concentration of 500 µg/ml. Store at 4°C.

G. Aureobasidin A Working ConcentrationAdd just 70 µl of the Aureobasidin A stock solution to 500 ml of dropout agar media for yeast two-hybrid screening, yielding a final concentration of 70 ng/ml.

H. X-a-Gal Stock Solution RecipeDissolve X-a-Gal (Cat. No. 630463) at 20 mg/ml in dimethylformamide (DMF). Store X-a-Gal solutions at –20°C in the dark.

I. X-a-Gal Working ConcentrationAdd 1 ml of the X-a-Gal stock solution to 500 ml dropout media for yeast two-hybrid screening.



40

Appendix D: Yeast Growth Media & Supplements continuedJ. SDO Agar Plates

Single dropout (–Trp) or (–Leu) media is prepared as follows:

Prepare 500 ml agar media using SD/–Trp (or SD/–Leu) with Agar, autoclave, and cool to 55–60°C in a •waterbath.

Mix, pour immediately, and allow to dry.•

K. SDO/X Agar Plates

Single dropout (–Trp) media containing 40 µg/ml X-a-Gal is prepared as follows:

Prepare 500 ml agar media using SD/–Trp with Agar, autoclave, and cool to 55–60°C in a waterbath.•

Add 1 ml of X-• a-Gal Stock Solution.


L. SDO/X/A Agar Plates

Single dropout (–Trp) media containing 40 µg/ml X-a-Gal and 70 ng/ml Aureobasidin A is prepared as follows:

Prepare 500 ml agar media using SD/–Trp with Agar, autoclave, and cool to 55–60°C in a waterbath.•


Add 70 µl of Aureobasidin A stock solution.•


M. DDO Agar Plates

Double dropout media is prepared as follows:

Prepare 500 ml agar media using SD–Leu/–Trp with Agar, autoclave, and cool to 55–60°C in a waterbath.•


N. DDO/X Agar Plates

Double dropout media containing 40 µg/ml X-a-Gal is prepared as follows:




O. DDO/X/A Agar Plates

Double dropout media containing 40 µg/ml X-a-Gal and 70 ng/ml Aureobasidin A is prepared as follows:






41


Appendix D: Yeast Growth Media & Supplements continuedP. QDO/X Agar Plates

Quadruple dropout media containing 40 µg/ml X-a-Gal is prepared as follows:

Prepare 500 ml agar media using SD/–Ade/–His/–Leu/–Trp with Agar, autoclave, and cool to 55–60°C in a •waterbath.



Q. QDO/X/A Agar Plates

Quadruple dropout media containing 40 µg/ml X-a-Gal and 70 ng/ml Aureobasidin A is prepared as follows:

Prepare 500 ml agar media using SD/–Ade/–His/–Leu/–Trp with Agar, autoclave, and cool to 55–60°C in a •waterbath.




R. Kanamycin Supplement to Yeast MediaKanamycin sulfate can be added to all yeast media at a final concentration of 50 µg/ml to stop bacterial contamination. Please note that kanamycin does not stop contaminating fungal growth, so proper sterile technique must still be used. Also note that this antibiotic does not select for any plasmids in yeast.



42

Notes

Notice to Purchaser

Aureobasidin Resistance Gene

This product is covered by the claims of U.S. Patent No. 6,015,689 and its foreign counterpart patent claims owned by Takara Bio Inc. Rights to use this product are limited to research only. Use of this product for purposes other than research may require a license, and inquiry concerning such commercial use should be directed to Takara Bio Inc., Seta 3-4-1, Otsu, Shiga 520-2193, Japan.

Clontech products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories, Inc.

Matchmaker™ Two-Hybrid System

Practice of the two-hybrid system is covered by U.S. Patent Nos. 5,283,173, 5,468,614, and 5,667,973 assigned to the Research Foundation of the State University of New York. Purchase of any Clontech two-hybrid reagent does not imply or convey a license to practice the two-hybrid system covered by these patents. Commercial entities purchasing these reagents must obtain a license from the Research Foundation of the State University of New York before using them. Clontech is required by its licensing agreement to submit a report of all purchasers of two-hybrid reagents to SUNY Stony Brook. Please contact the Office of Technology Licensing & Industry Relations at SUNY Stony Brook for license information (Tel: 631.632.9009; Fax: 631.632.1505).

Reverse Two-Hybrid Technology

This product is sold under license from Invitrogen IP Holdings, Inc, Carlsbad, CA, for use only in research conducted by the buyer of the product. The buyer is not authorized to use this product or its components for any therapeutic, diagnostic or prophylactic purposes and is not authorized to use this product or its components for any commercial purpose. The buyer is not authorized to sell this product or its components, is not authorized to sell any products made using this product or its components, and is not authorized to perform a service with this product or its components.

DH5a™ is a trademark of Invitrogen Corporation.

Parafilm® is a registered trademark of Pechiney Plastic Packaging, Inc..

Clontech, the Clontech logo and all other trademarks are the property of Clontech Laboratories, Inc. unless otherwise noted. Clontech is a Takara Bio Company. ©2008

Documents

Matchmaker™ Gold Yeast Two Hybrid System User Manual2].pdf · Matchmaker™Gold Yeast Two-Hybrid System User Manual Protocol No. PT4084-1 Clontech Laboratories, Inc. Version No